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A Patient-derived In Vitro - In Vivo Preclinical Model of Renal Cell Carcinoma

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ABSTRACT: Purpose: Authentic preclinical models of renal cell carcinoma (RCC) are lacking. We aimed to establish and characterize primary RCC cultures and demonstrate the feasibility of evaluating drug responses in vitro and in vivo. Materials and Methods: Previously published methodology, with minor modifications, was used to establish, cryopreserve, and serially passage RCC cells from nephrectomy and tumorgraft specimens. Cells were characterized for immuno- and molecular phenotype by immunochemistry, DNA sequencing and gene expression profiling. Tumorigenic potential was evaluated by implanting cells under the renal capsule of immunocompromised mice. The ability to monitor xenograft growth by magnetic resonance imaging (MRI) was investigated. Responses to a tyrosine kinase inhibitor (TKI) and an mTOR inhibitor were measured. Results: Primary cultures were successfully established from 11 clear cell and 1 chromophobe RCC, cryopreserved and serially passaged. Retention of immuno- and molecular phenotypes was demonstrated. Cultured cells formed xenografts in mice that could be measured by MRI. Patient-specific responses to drugs were observed in vitro and response to an TKI was confirmed in vivo. Conclusions: Our study is the first to show the derivation of primary cultures from RCC tumorgrafts, and to demonstrate the ability of primary RCC cultures to generate xenografts in mice. Our results suggest the feasibility of establishing large, well-annotated banks of RCC primary cultures for high-throughput drug screening in vitro and validation in vivo, providing a versatile platform together with xenografts and patient-derived precision-cut tissue slice tumorgrafts we developed previously for precilinical studies of RCC. Microarray analyses were performed to compare the gene expression profile of one of the primary cultures (case 7) to its parental tumor and tissue slice tumorgrafts in the study. Tissue slice grafts and parent tumors were preserved in Allprotect tissue reagent (Qiagen, Valencia, CA) at -20°C prior to RNA extraction using an AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Valencia, CA). The quality of RNA was determined using an Agilent 2100 Bioanalyzer (Agilent Biotechnologies, Santa Clara, CA). Microarray hybridization was performed using Illumina Human HT-12 v4 Beadchips (Illumina Inc., San Diego, CA) according to the manufacturer’s directions. Expression data was rank invariant normalized using BeadStudio software (Illumina Inc.).

Project description:Borna Disease Virus (BDV) is a neurotropic virus that persistently infects neurons in the central nervous system of various hosts, including rats. Although BDV is known to be an IFN sensitive virus, determination of the cellular mRNA transcript levels revealed the induction of IFN-stimulated genes in organotypic rat hippocampus slice cultures, raising the question how BDV evades this innate immune response. Using rat Mx protein as a specific marker for IFN-induced gene products, we could show that neurons lack detectable levels of Mx in these BDV infected cultures, whereas astrocytes and microglial cells were Mx positive. Neurons remained Mx negative after treatment of uninfected hippocampus cultures as well as primary dissociated neuronal cultures with high concentrations of IFN-α. This non-responsiveness correlated with a lack of a detectable nuclear translocation of pSTAT1 in rat neurons. Consistently, IFN treatment of BDV-infected rat neurons did not prevent the establishment of a viral persistence in the neuronal tissue. However, IFN treatment efficiently prevented vesicular stomatitis virus (VSV) replication, indicating that these cells can mount a weak innate immune response. In contrast, IFN treatment of mouse neurons resulted in the upregulation of Mx1 proteins and inhibition of BDV replication, indicating species-specific differences in the IFN response in neurons between mice and rats. Rat neurons may therefore represent the ideal cell type for BDV to evade the innate immune in the central nervous system. To determine the mRNA levels in the hippocampal slice cultures after BDV infection in Sprague Dawley and Lewis rats Hippocampal slice cultures were prepared from newborn Lewis and SD rats (P0-P2). Half of the samples remained uninfected, whereas the other half was infected with 1000 focus forming units (FFU) of BDV strain He/80. Total RNA from a pool of 12 slice cultures was prepared

Project description:Renal cell carcinoma (RCC) exhibits some unusual features and genes commonly mutated in cancer are rarely mutated in clear-cell RCC (ccRCC), the most common type. The most prevalent genetic alteration in ccRCC is the inactivation of the tumor suppressor gene VHL. Using whole-genome and exome sequencing we discovered BAP1 as a novel tumor suppressor in ccRCC that shows little overlap with mutations in PBRM1, another recent tumor suppressor. Whereas VHL was mutated in 81% of the patients (142/176), PBRM1 was lost in 58% and BAP1 in 15% of the patients analyzed. All these tumor suppressor genes are located in chromosome 3p, which is partially or completely lost in most ccRCC patients. However, BAP1 but not PBRM1 loss was associated with higher Fuhrman grade and, therefore, poorer outcome. Xenograft tumors (tumorgrafts) implanted orthotopically in mice retained >92% of mutations and exhibited similar DNA copy number alterations to corresponding primary tumors. Thus, after inactivation of VHL, the acquisition of a mutation in BAP1 or PBRM1 defines a different program that might alter the fate of the patient. Our results establish the foundation for an integrated pathological and molecular genetic classification of about 70% of ccRCC patients, paving the way for subtype-specific treatments exploiting genetic vulnerabilities. Overall design: The genomic DNA of clear-cell renal cell carcinoma (ccRCC) primary tumors, tumors growing in immunodeficient mice (tumorgrafts), and normal samples were labeled and hybridized to Affymetrix SNP arrays 6.0.

Project description:Renal cell carcinoma (RCC) exhibits some unusual features and genes commonly mutated in cancer are rarely mutated in clear-cell RCC (ccRCC), the most common type. The most prevalent genetic alteration in ccRCC is the inactivation of the tumor suppressor gene VHL. Using whole-genome and exome sequencing we discovered BAP1 as a novel tumor suppressor in ccRCC that shows little overlap with mutations in PBRM1, another recent tumor suppressor. Whereas VHL was mutated in 81% of the patients (142/176), PBRM1 was lost in 58% and BAP1 in 15% of the patients analyzed. All these tumor suppressor genes are located in chromosome 3p, which is partially or completely lost in most ccRCC patients. However, BAP1 but not PBRM1 loss was associated with higher Fuhrman grade and, therefore, poorer outcome. Xenograft tumors (tumorgrafts) implanted orthotopically in mice exhibited similar gene expression profiling to corresponding primary tumors. Gene expression profiling of tumors and tumorgrafts displayed different signatures for BAP1- and PBRM1-deficient samples. Thus, after inactivation of VHL, the acquisition of a mutation in BAP1 or PBRM1 defines a different program that might alter the fate of the patient. Our results establish the foundation for an integrated pathological and molecular genetic classification of about 70% of ccRCC patients, paving the way for subtype-specific treatments exploiting genetic vulnerabilities. The RNA of clear-cell renal cell carcinoma (ccRCC) primary tumors, tumors growing in immunodeficient mice (tumorgrafts), and normal kidney cortices were labeled and hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays.

Project description:The neurite outgrowth inhibitory myelin protein Nogo-A has been well studied in the context of central nervous system (CNS) injury and disease. We studied the effects of the application of neutralizing anti-Nogo-A antibodies (11C7 and 7B12) in intact CNS tissue in vitro using rat organotypic hippocampal slice cultures. This study had the purpose of elucidating the role of Nogo-A in the adult intact CNS and determining the consequences of its neutralization through antibody application. In vitro cultures treated with anti-Nogo-A antibody showed an elicited growth response. The results also gave indications that hippocampal circuitry might be altered due to the regulation at the synaptic and neurotransmission level. Experiment Overall Design: Nogo-A function in the intact CNS tissue is not well known, but its neutralization in vivo produced a transitory growth response of Purkinje axons and of the corticospinal tract in intact adult rats (Buffo et al., 2000; Bareyre et al., 2002; Gianola et al., 2003). Nogo-A is relatively highly expressed in oligodendrocytes and some neurons of the hippocampus (Huber et al., 2002; Meier et al., 2003; Gil et al., 2006; Trifunovski et al., 2006). Organotypic hippocampal slice cultures are a good in vitro model to study hippocampal function and structure (Stoppini et al., 1991; 1993; Bahr, 1995; Gahwiler et al., 1997; Hakkoum et al., 2006). They mature in vitro and retain many in vivo features from a structural and functional perspective. We chose this model to study the effects of acute Nogo-A neutralization, using two function blocking monoclonal antibodies, 11C7 and 7B12 (Oertle et al., 2003; Wiessner et al., 2003; Liebscher et al., 2005), exclusively targeted against the Nogo-A specific region. Hippocampal slices from P7 Wistar rats were cultured for 21 DIV. Control untreated cultures where cultured for additional 5days for a total of 26DIV, while control IgG, and 11C7 and 7B12 were added to 21DIV cultures which were then further cultured for 5days, changing medium every 2 days. All the conditions were repeated in triplicates with separate cultures from different animals. For each condition and experimental replicate 24 cultures were pooled together before being processed for RNA extraction. Data analysis was performed by GeneSpring 7.2 (Silicon Genetics, Agilent, CA, US) comparing 11C7 and 7B12 treated samples versus Not treated and IgG treated, as controls. A present call filter (2 out of 3 present calls in at least one out of the 3 experimental replicates) was applied. Normalization was run per chip as well as per gene to the median of the control replicates. Data were statistical restricted through a 1-way Anova (p≤0.05). A final threshold of ≥1.2 fold of increase or decrease in the expression level of each single transcript was applied. Regulated transcripts have been assigned to functional categories according to GeneOntology as well as literature and database mining (Pubmed; Bioinformatics Harvester EMBL Heidelberg; Rat Genome Database).

Project description:Renal cell carcinoma (RCC) exhibits some unusual features and genes commonly mutated in cancer are rarely mutated in clear-cell RCC (ccRCC), the most common type. The most prevalent genetic alteration in ccRCC is the inactivation of the tumor suppressor gene VHL. Using whole-genome and exome sequencing we discovered BAP1 as a novel tumor suppressor in ccRCC that shows little overlap with mutations in PBRM1, another recent tumor suppressor. Whereas VHL was mutated in 81% of the patients (142/176), PBRM1 was lost in 58% and BAP1 in 15% of the patients analyzed. All these tumor suppressor genes are located in chromosome 3p, which is partially or completely lost in most ccRCC patients. However, BAP1 but not PBRM1 loss was associated with higher Fuhrman grade and, therefore, poorer outcome. Xenograft tumors (tumorgrafts) implanted orthotopically in mice retained >92% of mutations and exhibited similar DNA copy number alterations to corresponding primary tumors. Thus, after inactivation of VHL, the acquisition of a mutation in BAP1 or PBRM1 defines a different program that might alter the fate of the patient. Our results establish the foundation for an integrated pathological and molecular genetic classification of about 70% of ccRCC patients, paving the way for subtype-specific treatments exploiting genetic vulnerabilities. The genomic DNA of clear-cell renal cell carcinoma (ccRCC) primary tumors, tumors growing in immunodeficient mice (tumorgrafts), and normal samples were labeled and hybridized to Affymetrix SNP arrays 6.0.

Project description:Brain slice cultures offer advantages over other in vitro methods, as they mimic numerous in vivo aspects. For most purposes, slices of the developing brain, termed organotypic slice cultures , preserve a high degree of cellular differentiation and tissue organization. The entorhino-hippocampal connection (EHP) is the main entrance of information to the hippocampus proper and the dentate gyrus. Also it has some specific features that make them particularly interesting in studies of axonal regeneration: (i) the culture method obviates the need for extensive animal surgery and requires less time than other in vivo approaches; (ii) the EHP is reproduced easily in vitro in cultures with a degree of laminar specificity similar to that found in vivo; (iii) the EHP is myelinated both in vitro and in vivo; and (iv) most of the cellular and molecular barriers to axon regeneration are present after the axotomy of the EHP in vitro. Altogether this model is useful to evaluate axon regeneration and putative estrategies to promote axonal growth. Keywords: organotypic slice cultures; axonal lession; gene expression To evaluate the genes whose transcription was regulated after 1, 3 and 7 days after EHP (Entorhino-Hippocampal Pathway) axotomy, RNA samples were analyzed with Agilent whole genome rat long oligonucleotide (44 K base) probe based microarrays.

Project description:Patients with metastatic renal cell carcinoma (RCC) have a life expectancy of 6 months to 1 year. The deadly nature of RCC compared to other tumors that metastasize to bone, such as prostate cancer (PC), is associated with extensive arteriogenesis that requires recruitment of muscle progenitor cells to form the vascular smooth muscle around these large vessels. To identify potential genes that are involved in RCC recruitment of muscle progenitor cells we performed a microarray analysis to evaluate the global gene expression of human RCC (786-O) cells that form these large vessels in murine xenografts, versus human PC (PC3) that do not form these large vessels during osteolytic bone metastasis in mice (Xie C, et al. J Orthop Res. 2011;30(2):325-33). To assess changes in gene expression that occur when tumor cells interact with muscle progenitor cells, primary myoblast isolated from 5-day-old C57BL/6-Tg GFP neonatal mouse limbs were co-cultured with RCC or PC cells. Overall design: The experiment contained five groups: [Group 1] pure 786-O cells; [Group 2] pure PC3 cells; [Group 3] 1:1 co-culture of myoblast with 786-O cells; [Group 4] 1:1 co-culture of myoblasts with PC. All cultures were plated at 80% confluence, and the cells were harvested 12 hours later to isolate total RNA.